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...standardizing the process. When we started, too much time for this project was spent selecting guides, designing... CrispyCrunch While CrispyCrunch may evolve over time, you can depend on it to stick to a few guiding ...Furthermore, we did not find any tools which were optimized for HDR, except for TagIn. HDR requires the additional...enabled it to return guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide...insert, the more poorly the guide performs HDR. Optimal mutation in-depth One the most sophisticated features...mind, we implemented the following algorithm for optimal mutation in CrispyCrunch: If the guide PAM resides...

...allowing you to spend less time researching plasmid basic features and spend more time developing the clever...as a means of detection; they are useful almost anytime you want to express multiple genes in the same ...the genes expressed from the same cassette is sometimes advantageous, particularly when only a portion... be feasible to express more than two genes at a time using IRES elements. Further, scientists have reported...Likely the choice of specific 2A peptide will ultimately depend on a number of factors such as cell type...

... a 2D barcoded tube (see next section) and every time something is removed from this tube, the barcode...up samples as we transform dozens of samples at a time.   Not everything that is received is immediately... Most of our samples (98.2%) transform the first time and can be stored right away. Every week, the lab...better for tubes that are meant to be read one at a time. 1D barcodes can be purchased from many companies...prevents us Addgenies from wasting hours of valuable time searching for samples and keeps us from discarding... samples get lost during their journey. Take the time to track your samples today and never lose a sample...

...accuracy. Conversely, you can only run one sample at a time, which can become costly and impractical for whole-genome...repetitive regions. Short-read sequencing can also be time-consuming, depending on the library prep and instrumentation...anywhere from under an hour to 72 hours, with real-time data output. This method is great for resolving ...quality control. This gives us a faster turnaround time than short-read sequencing, as well as the ability... containing cassettes of the same gene multiple times.  Bioinformatics at Addgene After a run on our Illumina... QC team members. This process saves the QC team time, but does not leave much flexibility for discrepancies...

...repository through our Hot Plasmids articles. Today, it's time for another edition of Hot Plasmids!  Here's what... size-limited AAV delivery. PE6c and PE6d are optimized for dual-AAV systems. These variants use different... tagged iPAK4 from different promoters, specific time points appear as fluorescent bands in the fiber,...tape” recording. The authors created externally-timed bands of color with HaloTag-iPAK4 by applying different... accurate to within 40 minutes. Figure 4: Time-resolved protein ticker tape. A) Design of iPAK4...iPAK4 plasmids here! Lin D., Li X., et al. (2023). Time-tagged ticker tapes for intracellular recordings...

...and translating a protein fluorophore also takes time - bypassing translation altogether with an oligonucleotide-only...oligonucleotide-only sensor could save precious minutes in a time sensitive detection assay. In 1999, Grate and Wilson... a non-protein based visualization technique. Ultimately, MG proved to be too toxic for many in vivo applications...aptamer, called Spinach, required longer exposure time than its protein equivalent and was less stable ...throughput assays with microarrays and biochips. Ultimately, there are just some applications that lend themselves...imaging of RNA processing using broccoli reveals optimal aptamer expression strategies.” Chem Biol. 2015...

... lot of great output in a short amount of time can sometimes become cauldrons on the verge of boiling....inform a go/no-go decision in a very short amount of time. Such a decision determines whether the client invests...achievable tasks, and then executing on them in a timely fashion while allowing for creative solutions to...services that help diabetic patients manage doses and timings of their drug treatments. The process of rising...goal posts” as a part of a case, consulting can sometimes be challenging. The fast paced nature of projects...

...with proper protein folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common...reductase to faciliate proper folding; may increase multimer formation Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII ...as IPTG is added to the growth medium. Induction timing is important, as you typically want to make sure...reaching the desired density within a reasonable time-frame. For these cases, some strains carry an additional...experiment to test variables such as temperature, time, and media conditions. Many recombinant proteins...many proteins, a rich media such as TB or 2XYT is optimal because of the high cell-density they support; ...

...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell needed...of a histogram, which graphically represents the time and intensity of the cell’s illumination (see Figure...maximum signal strength, while W results from the time that the cell spent passing through the laser beam...clearly distinct positive and negative populations. Oftentimes, you’ll be presented with a trend of two populations... With our first three gates out of the way, it’s time to move on to my experimentally-specific gates. ...

... Taking control of a RhoGEF is a great way to stimulate and study these physical processes. Joachim Goedhart...inhibit it. Restricting the effect to a specific time or location can be critical when, say, the gene ... protein’s function at a very specific place and time. Future studies with these CIB1-Rabs in other contexts...variants called SuperPLDs with 10, 30, 50, and 100 times the activity of the original, generated using directed...monitored by phosphatidic acid probe GFP-PASS. B) Time series of HEK 293T cells expressing plasma membrane-targeted.... D., & Goedhart, J. (2023). Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho ...

...share my experiences from my own job search and my time on faculty search committees. Having successfully...you think you can accomplish. No matter how many times I say this, candidates regularly try to convince...The second question is whether there is chalk. Sometimes. It can also be a slide deck, markers, etc. Chalk... them into simple goals/outcomes. This isn't the time to go into detail. Finally, state how successfully...the aims of a chalk talk. Questions take a lot of time to discuss and the questions are important. However...secretory proteins in the Endoplasmic Reticulum, optimization of fluorescent proteins,  and live cell imaging...

... to alleviate those differences, they had a hard time maintaining their friendships. I put “neutral” in...ability to remember the right stories at the right time. Like many of the posts in our Transferable Skills...address these types of harmful conflict early, because time frequently does not heal these types of wounds. ... know about those particular teams at that given time. I think about how previous iterations of those ...collaborating, and compromising. Though there may be a time and place for avoiding, competing, or accommodating...looking for a promotion, have some solid examples of times when you have used your empathy, listening skills...

...pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing is then...concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased...Wrenbeck et al for details on how nicking was optimized. How should I design my mutagenic primers? The...orientation of the BbcVI restrict site matter? Most of the time no, but there are 2 instances where it does. 1) ...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site or...Stapleton, Adebola Adeniran, Keith E J Tyo, and Timothy A. Whitehead. "Plasmid-based one-pot saturation...

...incredibly fast protocol, with only 5 hours incubation time needed for a single round of editing, and a subsequent...other advantages including shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces...identification of a new CRISPRi tool, they also optimized the system by modulating the PAM to further tune...-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32:1146–1150 . https://doi....RJ, Mimee M, Lu TK (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases... Sommer MOA, Nielsen AT (2016) CRMAGE: CRISPR Optimized MAGE Recombineering. Sci Rep 6: . https://doi....difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials. mBio 11: . https://doi.org/10.1128/mbio.00019...

...culturist has to deal with this problem. It is estimated that mycoplasma is responsible for up to 60% of...air if the cell line remains exposed for a longer time. The aerosols are invisible to naked eyes and hence...Mycoplasmas can remain in the dry state for a longer time. When they come in contact with a nutrient source...continuous culture for at least two weeks to give time for low levels of contaminations to grow. For testing...products or reagents that have been stored for a longer time. Use fresh media as much as possible and the ones...ways to rescue the cultures. Elimination is a very time-consuming process and increases the risk of secondary...

...will influence the stability of your sample over time and determine which experiments you can effectively... solution is fundamental, and it will impact the time you will be able to store your sample without having...preserving the stability of your preparation for a long time. Tris buffer controls the pH, while the EDTA chelates...stability during storage over prolonged periods of time, especially at room temperature or at 4°C. Storing...better than water in these conditions.  The next time you do a plasmid prep, plan in detail before you...

... do my internship in the daytime.  Getting into the office for the first time, being introduced to all...on a time crunch and knew that if we were to restart the procedure we would not have enough time. During...I got from the La Vida Scholars tour. After some time had passed, I reached out to Amy with exciting news...

...Tm and primer dimer estimation, and other primer characteristics. NEB Data-optimized Assembly Design (DAD...post-translational modifications and polymorphisms Codon optimization tool: To access this tool, set up an IDT account...Sequence tool.   DNA sequence manipulation tools  Sometimes DNA sequences need to be “cleaned up” or changed...Sharing your lab protocols using apps to save you time and track your experiments  Resources on Addgene.org...

...targeted region, as shown below.  To identify optimal nickase designs, IDT scientists Mollie Schubert...and Yan next examined a situation thought to be suboptimal for HDR. At the AAVS1 locus, the two nearest ...13 nt away from the target site rather than the optimal <10 nt distance. Employing two gRNA sites that ...’s best advice: Use a PAM-out configuration Optimize your spacing D10A: nick sites separated by 37...in this post were adapted from an IDT webinar: Optimized methods to use Cas9 nickases in genome editing...

...at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a brief comparison...Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70 °C Genome Editing...have increased thermostability also had a longer lifetime in human blood. To test if this was also true ...without the need to grow the organisms at lower than optimal temperatures. GeoCas9’s thermostability could also...Lucas, et al. "A thermostable Cas9 with increased lifetime in human plasma." bioRxiv (2017): 138867. Additional...